Project Summary Alzheimer's disease (AD) is a neurodegenerative disorder with no effective cure. Genome-wide association studies (GWAS) have identified a large number of genetic variants, mostly in the form of single nucleotide polymorphisms (SNPs), that are associated with AD. Identification of the causative SNPs among the AD- associated genetic variants will provide important insights into etiology of the disease and therapeutic targets. Expression quantitative trait loci (eQTLs) studies can identify SNPs that are likely to affect downstream gene expression. However, this approach cannot provide any information on SNP-binding proteins. Intersecting GWAS SNPs with transcription factor (TF) binding sites by ChIP-seq is a useful approach to identify functional SNPs and their interacting TFs but requires a priori knowledge of the relevant TFs. Another option is to identify differential protein binding to a SNP-carrying DNA fragment using pull down-coupled mass spectrometry; however, it is difficult to scale up to identify differential binding proteins for a large number of SNPs. In this proposal, we propose to implement a Proteome-Wide Analysis of disease-associated SNPs (PWAS) study on non-protein coding regions to identify allele-specific protein-DNA/RNA interactions and alteration of regulatory activity in AD. This is based on our hypothesis that functional DNA/RNA SNPs likely execute their function via allele-specific interactions with proteins. We will survey the entire human TF and RNA-binding protein repertoires with SNP-carrying DNA and RNA probes using a protein array-based approach. This assay is also extremely high-throughput because >20,000 human proteins can be simultaneously surveyed for each probe. Identified allele-specific protein-DNA and -RNA interactions will be prioritized using a series of bioinformatics analyses and validated using human cells differentiated from induced pluripotent stem cells. To achieve our goals, we propose four aims. Aim 1: Determine DNA allele-specific SNP-TF interactome. We will perform protein-DNA interaction assay with 75 AD-associated SNPs validated by gel-shift assays. Aim 2: Identify RNA allele-specific SNP-protein interactome. We will focus on 75 SNPs located in 5'-UTR, 3'-UTR and intronic regions, which are likely to affect RNA splicing, mRNA stability, and protein translation. Aim 3: Assess the biological consequences of prioritized allele-specific interactions in AD relevant specific cell types. We will integrate the protein-DNA/RNA interactions with existing genomic datasets to prioritize a subset of allele- specific protein-DNA/RNA interactions for validation in human cell populations. Aim 4: Construct a PWAS browser for human diseases. We will develop a user-friendly PWAS browser to distribute the allele-specific protein-DNA/RNA interactions that are relevant to human diseases. The success of this project is expected to establish a powerful platform and provide a rich resource to rapidly identify functional SNPs and provide crucial biological insights into the molecular mechanisms underlying AD.